The accessory genes, vpr and vpx, are highly conserved across primate lentiviral lineages. Several genetically and functionally separable properties have been described for these accessory proteins. HIV-1 Vpr has been implicated in nuclear translocation of viral reverse transcription complexes. In addition, this protein causes a delay in the G2 phase of the cell cycle and has additionally been shown to associate with the DNA repair enzyme, uracil DNA glycosylase. These properties are segregated between Vpr and Vpx of HIV-2/SIVSM in that Vpr influences cell cycle progression and associates with UDG, whereas, Vpx promotes nuclear translocation of viral cDNA in nondividing macrophages. Studies completed in the previous funding period have underscored the notion that nuclear targeting activities of HIV-1 Vpr and its analogous activity in HIV-2/SIVSM Vpx have evolved to permit efficient virus replication in primary macrophages both in vitro and in vivo. Without information on functional domains in Vpr/Vpx proteins which regulate their activities is that mutations in activating Vpr and Vpx alleles may compromise possible additional as yet unidentified functions to more rigorously connect the replication defect in vivo imposed by the Vpx mutation with impaired nuclear import in nondividing macrophages would require the identification of defined mutants which specifically impair the nuclear import activity of these proteins. The object of this proposal is to more rigorously establish the contribution of nuclear transport activities of Vpr/Vpx proteins to primate lentiviral pathogenicity. Specifically, we propose to: Aim 1: Identify effector domains for nuclear localization of HIV-1 Vpr and SIVSM Vpx and for association of Vpr/Vpx with viral reverse transcription complexes. Aim 2: Characterize the pathway through which nuclear localization of Vpr/Vpx proteins is directed and identify the potential role of bridging proteins in the interaction of Vpr/Vpx with the nuclear import apparatus. Aim 3: Examine the phenotype of nuclear import mutants of HIV-1 and SIV in macrophage infection. Aim 4: Examine the relative in vivo fitness of SIVSM Vpx mutants which are impaired in nuclear import. It is expected that these studies will more fully define the contribution of nuclear import activities of Vpr and i Vpx proteins to virus replication in vitro and in vivo.